The normal development of red blood cells requires a coordinated synthesis of the hemoglobin (Hb) subunits, the α- and β-globins in the case of adult hemoglobin (Hb A). The α- and β-globin chains are encoded by genes on different chromosomes, 16 and 11 respectively, and their expression is controlled independently. In the normal red blood cell, slightly more α-chains than β-chains are produced. Unlike the β-hemoglobin chains (β-Hb) which are soluble and form homologous tetramers, the free α-hemoglobin chains (α-Hb) are highly instable, and when in excess, form precipitates and act as active oxidants causing apoptosis and inefficient erythropoiesis.
β-thalassemias are inherited autosomal recessive diseases characterised by a decrease or abolition of the normal β-globin chain synthesis inducing inefficient erythropoiesis (Weatherall, 2004). Consequences include anemia, of different severity according to the mutations involved, several other severe disorders due to the increase in medullar erythropoiesis, impaired statural growth and bone structure, accelerated iron turnover, and heme catabolism, and their own clinical consequences.
In 2002, the molecular chaperone of α-Hb, the <<Alpha-Hemoglobin Stabilizing Protein>> (AHSP) was reported (Kihm et al., 2002). This small protein of 102 amino-acids is present at a high level (0.1 mM) in human red blood cell precursors and its synthesis is under the control of GATA-1, a pivotal erythroid transcription factor. This protein is encoded by chromosome 16. Also, in contrast to most other molecular chaperones, which are widely expressed and relatively promiscuous with respect to substrate interactions, AHSP appears to be highly tissue and substrate specific. AHSP specifically binds to α-Hb to form a stable soluble heterodimer but not to the β-Hb or to tetrameric Hb A (Kihm et al., 2002; Gell et al., 2002). Hence the role of AHSP could be to prevent free α-Hb from aggregation until the encounter of β, δ or γ chains allows formation of the corresponding tetrameric Hbs. In the red blood cell of β-thalassemic patients, AHSP acts as a scavenger against the pool of free α-chain but may be overwhelmed by a defective production or level of availability of β-like chains. Thus, the free α-Hb monomers in the red cells overload the AHSP capacity and precipitate, damaging the cell and triggering cell apoptose. For β-thalassemic patients, the free α-chain pool in the red blood cell may thus reflect the severity of a β-thalassemia syndrome.
Based on AHSP identification, US Patent Application 2005/0028229 describes a method of diagnosing an AHSP-related disorder in a test subject such as β-thalassemia by determining the presence in a sample from said test subject of AHSP and, if present, determining the expression level.
However, currently the diagnosis of β-thalassemia is still based on the hematological parameters of the patients and the molecular diagnosis is obtained by PCR techniques. More than 200 different β-thalassemia mutations have now been characterized, the majority of which are point mutations or very short deletions/insertions. Most of these mutations are country or population-specific, and their distributions have now been determined for most at-risk populations. The strategy for identifying these mutations is usually based on the fact that most populations have just a few common mutations and a variable number of rare ones. The severity of the β-thalassemia depends mainly on the nature of the mutation. Two main classes of disorders are described, first the β0-thalassemia (β0-thal) in which no β chains are produced and second the β+-thalassemia (β+-thal) in which some normal β chains are synthesized. The clinical manifestations of β-thalassemia are extremely diverse ranging from severe anemia and transfusion-dependency to the asymptomatic state of β-thalassemia trait (Thein, 2005). More generally one may need to consider the overall imbalance between the α and β family of globin chains, in order to include the different stages of development; for example, the .beta. family includes the fetal (γ) and adult (β) chains. The great variability in the phenotypic expression of the .beta.-thalassemia also depends on association with some modifiers of Hb synthesis which may modify the .alpha. biosynthetic ratio between cluster β and α-globin.
Thus, the central pathological mechanism of β-thalassemias is the imbalance between the synthesis of the alpha and beta (γ+β) family of globin chains (Weatherall and Clegg, 2001) and the severity of this disease is directly correlated with the degree of the globin chain imbalance. The in vitro study of synthesis of the α- and γ+β-globin chains of Hb from peripheral blood reticulocytes highlighted in 1965 this imbalance of globin synthesis in thalassemia (Weatherall et al., 1965). Many subsequent studies have reported the same usefulness of measuring the imbalance of globin chain synthesis from β-thalassemic reticulocytes, but all the different laboratory methods are based on the incorporation of a radioactive amino acid in the subunit biosynthesis from peripheral blood (Kim et al., 1977). Such a method could certainly not be considered as routine laboratory practice.
It results that there is currently no simple and rapid test to evaluate this parameter. In routine hematologic examinations, the excess of free α-Hb may be distinguished in cytology by the presence of inclusion bodies corresponding to the denatured and precipitated α-Hb but this approach is not specific of the α-Hb and only qualitative. It is cumbersome, expensive and time consuming procedure.
Indeed, the only technique to quantify the relative excess of free α-Hb is to carry out globin biosynthesis in vitro in the presence of a radioactive amino-acid. This technology has been used in research laboratories in the 1970s, 1980s and the measurement of the amount of radioactivity in different collected globin fractions allows a determination of the α/β chain synthesis ratio. This characterization method is less and less in use, even in research laboratories, because of the use of radioactivity.
Furthermore, until very recently, it was considered that was impossible to detect or quantify free α-Hb since the excess α-Hb either precipitates in erythroid precursors in the bone marrow (resulting in ineffective erythropoiesis), causing their premature destruction, and although partly proteolyzed binds to the cell membrane of adult erythroid cells, leading to their hemolysis and promoting apoptosis (Bank, 2007 and Yu et al., 2007).
Therefore, there is a need for a method for easily diagnosing and/or staging a hemoglobin-related disorder such as β-thalassemias carried out without using molecular or radioactive technique.